Mbnl2 loss alters novel context processing and impairs object recognition memory

Abstract

Patients with myotonic dystrophy type I (DM1) demonstrate visuospatial dysfunction and impaired performance in tasks requiring recognition or memory of figures and objects. In DM1, CUG expansion RNAs inactivate the muscleblind-like (MBNL) proteins. We show that constitutive Mbnl2 inactivation in Mbnl2^ΔE2/ΔE2 mice selectively impairs object recognition memory in the novel object recognition test. When exploring the context of a novel arena in which the objects are later encountered, the Mbnl2^ΔE2/ΔE2 dorsal hippocampus responds with a lack of enrichment for learning and memory-related pathways, mounting instead transcriptome alterations predicted to impair growth and neuron viability. In Mbnl2^ΔE2/ΔE2 mice, saturation effects may prevent deployment of a functionally relevant transcriptome response during novel context exploration. Post-novel context exploration alterations in genes implicated in tauopathy and dementia are observed in the Mbnl2^ΔE2/ΔE2 dorsal hippocampus. Thus, MBNL2 inactivation in patients with DM1 may alter novel context processing in the dorsal hippocampus and impair object recognition memory.

Keywords: Biological sciences; Molecular biology; Molecular mechanism of gene regulation; Neuroscience; Transcriptomics.

Graphical abstract

Figure 1

Mbnl2^ΔE2/ΔE2 mice demonstrate impaired object recognition memory Fear conditioning A–D: A: Percent freezing during the context test. (B) Percent freezing during the generalization test. (C) Percent freezing in the tone test: raw freezing scores. (D) Percent freezing in the tone test: covaried by baseline level of freezing. Covariance is computed at the group level so individual data points cannot be plotted. NORT E–H: (E and F). Locomotor activity in the open field on days 1 and 2. (G and H) Preference for investigation of the novel object on the test day. For all behavioral tests, the numbers of male mice 4–5 months of age used were Mbnl2^+/+ = 9; Mbnl2^ΔE2/ΔE2 = 10. WT: Mbnl2^+/+; KO: Mbnl2^ΔE2/ΔE2. Means +SEM are shown. In all cases, ∗ and ∗∗ indicate levels of significance where p < 0.05 and p < 0.01, respectively.

Figure 2

Mbnl2^ΔE2/ΔE2 dorsal hippocampus initiates an aberrant transcriptome response when exploring the context of a novel arena (A) Volcano plots of transcriptome differences in the dorsal hippocampi of trained versus house control Mbnl2^+/+ mice (Ai), trained versus house control Mbnl2^ΔE2/ΔE2 mice (Aii), house control Mbnl2^ΔE2/ΔE2 mice versus house control Mbnl2^+/+ mice (Aiii), and trained Mbnl2^ΔE2/ΔE2 mice versus trained Mbnl2^+/+ mice (Aiv) are shown. In all cases, the log2 fold change and the corresponding p value are plotted on the x axis and y axis, respectively. Significant gene alterations (p < 0.05) are shown in red and non-significant alterations are shown in black. All corresponding histograms are shown in Figure S1. Data are tabulated in Tables S1, S2, S3, S9, and S11. (B) Heatmaps show the range and distribution of genes with significant p values (p < 0.05) and log2 fold change >1.5 (<-1.5). Dorsal hippocampi from trained versus house control Mbnl2^+/+ mice (Bi), trained versus house control Mbnl2^ΔE2/ΔE2 mice (Bii), house control Mbnl2^ΔE2/ΔE2 mice versus house control Mbnl2^+/+ mice (Biii), and trained Mbnl2^ΔE2/ΔE2 mice versus trained Mbnl2^+/+ mice (Biv) are shown. Data are tabulated in Table S4. (C) qPCR analysis of steady-state mRNA level alterations in the dorsal hippocampi of house control and trained Mbnl2^+/+ and Mbnl2^ΔE2/ΔE2 mice are shown. Dorsal hippocampi were derived from n = 3 mice/genotype/condition. Means +SEM are shown. WT: Mbnl2^+/+, KO: Mbnl2^ΔE2/ΔE2, HC: house control, TR: trained. In all cases, ∗, ∗∗, and ∗∗∗ indicate levels of significance where p < 0.05, p < 0.01 and p < 0.001 respectively; NS: not significant.

Figure 3

Mbnl2^ΔE2/ΔE2 dorsal hippocampus responds to the context of a novel arena with a lack of enrichment for cellular and functional pathways implicated in learning and memory (A and B) Scatterplots of log2(TR/HC) in WT mice (Mbnl^+/+ novel context exploration) versus log2(TR/HC) in KO mice (Mbnl2^ΔE2/ΔE2 novel context exploration) are shown. Genes significantly altered in both cases are indicated in black. Genes that are significantly altered only with Mbnl^+/+ but not with Mbnl2^ΔE2/ΔE2 novel context exploration are shown in red and assigned their true values in Panel a, and genes that are significantly altered only with Mbnl2^ΔE2/ΔE2 but not with Mbnl^+/+ novel context exploration are indicated in blue and assigned their true values in Panel B. WT: Mbnl2^+/+, KO: Mbnl2^ΔE2/ΔE2, HC: house control, TR: trained. (C) All cellular functions showing significant non-random enrichment only with Mbnl^+/+ but not with Mbnl2^ΔE2/ΔE2 novel context exploration, with both Mbnl^+/+ and Mbnl2^ΔE2/ΔE2 novel context exploration and only with Mbnl2^ΔE2/ΔE2 but not with Mbnl^+/+ novel context exploration are shown. IPA activation Z score < −2 or >2 and q values (B–H multiple testing correction p values) for the cellular functions are indicated. The number of genes for each cellular function is shown in blue circles. Individual gene alterations for the cellular functions in Panel c are shown in Data S1–S3 and in Tables S5, S6, S7, and S8. The IPA prediction legend is shown in Figure S2.

Figure 4

The transcriptome of the house control Mbnl2^ΔE2/ΔE2 dorsal hippocampus overlaps with the transcriptome alterations occurring in the Mbnl2^+/+ dorsal hippocampus with novel context exploration (A) Shared cellular functions showing significant non-random enrichment in house control Mbnl2^ΔE2/ΔE2 mice (house control Mbnl2^ΔE2/ΔE2 versus house control Mbnl2^+/+ mice) and in Mbnl^+/+ mice with novel context exploration (trained versus house control Mbnl^+/+ mice) are shown. IPA activation Z score < −2 or >2 and q values (B–H multiple testing correction p values) for the cellular functions are indicated. The number of genes for each cellular function of the house control Mbnl2^ΔE2/ΔE2 mice is shown in gray circles. The numbers of shared gene alterations for each cellular function in Mbnl^+/+ novel context exploration and in house control Mbnl2^ΔE2/ΔE2 mice are indicated above the gray pies. WT: Mbnl2^+/+, KO: Mbnl2^ΔE2/ΔE2, HC: house control, TR: trained. (B and C) Individual gene alterations for increased synaptic transmission (B) and increased transport of synaptic vesicles (C) occurring with novel context exploration in the dorsal hippocampus of Mbnl^+/+ mice are shown. Genes comparably altered in the house control Mbnl2^ΔE2/ΔE2 dorsal hippocampus are ringed in red and genes that are inversely altered are ringed in blue. Individual gene alterations for the cellular functions in Panel a are shown in Data S4 and S5 and in Tables S2, S9, and S10. The IPA prediction legend is shown in Figure S2.

Figure 5

Post novel context exploration, the Mbnl2^ΔE2/ΔE2 dorsal hippocampus shows changes in genes implicated in tauopathy and dementia (A–C) q values (B-H multiple testing correction p values) of genes implicated in tauopathy (A) in the dorsal hippocampus of house control Mbnl2^ΔE2/ΔE2mice (house control Mbnl2^ΔE2/ΔE2 versus house control Mbnl2^+/+ mice; indicated as KO/WT in HC) and q values of genes implicated in tauopathy (B) and dementia (C) in trained Mbnl2^ΔE2/ΔE2mice (trained Mbnl2^ΔE2/ΔE2 versus trained Mbnl2^+/+ mice; indicated as KO/WT in TR) are shown. In panels B and C, all genes that are comparably altered in the dorsal hippocampus of the house control Mbnl2^ΔE2/ΔE2 mice and the trained Mbnl2^ΔE2/ΔE2 mice are ringed in blue and genes that are altered significantly only in the dorsal hippocampus of trained Mbnl2^ΔE2/ΔE2mice are ringed in green. Individual gene alterations are shown in Tables S9, S11, and S12. The IPA prediction legend is shown in Figure S2.

Figure 6

Mbnl2 inactivation impairs novel context processing and object recognition memory